Antigen recognized by patients with antibody associated paraneoplastic sensory neuronopathy, DNA encoding same and uses thereof

ABSTRACT

An isolated nucleic acid sequence encoding Hu paraneoplastic antigenic polypeptide is provided by this invention. This invention also provides a purified Hu antigenic polypeptide and compositions containing the purified Hu antigenic polypeptide. Further provided by this invention is a monoclonal antibody directed to an epitope on the Hu paraneoplastic antigenic polypeptide. Compositions containing this monoclonal antibody also are provided by this invention. This invention also provides methods of diagnosis and treatment using the compositions described hereinabove.

This application is a continuation of U.S. Ser. No. 08/461,027, filedJun. 5, 1995, now U.S. Pat. No. 5,807,705, issued Sep. 15, 1998, whichis a divisional of U.S. Ser. No. 08/210,732, filed Mar. 18, 1994, nowU.S. Pat. No. 5,603,934, issued Feb. 18, 1997, which is a continuationof U.S. Ser. No. 07/665,959, filed Mar. 4, 1991, now abandoned, thecontents of which are hereby incorporated by reference into the presentapplication.

This invention was made in the course of work under Grant No. PD 359from the American Cancer Society. The United States government hascertain rights in this invention.

BACKGROUND OF THE INVENTION

Throughout this application various references are referred to withinparentheses. Disclosures of these publications in their entirety arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citations for these references maybe found at the end ofthis application, immediately preceding the claims.

Paraneoplastic sensory neuronopathy is a rare remote effect of cancercharacterized clinically and pathologically by neuronal loss andinflammatory infiltrates. (1, 6, 7) A high percentage of such patientsharbor a characteristic serum antibody which has been called anti-Hu.(4) These sera react with a group of proteins which have been found tobe specifically expressed in tumor tissue (usually small cell lungcancer) and brain. (8) This observation suggested a model for theneurological dysfunction in which an immune response primarily directedagainst a tumor antigen is misdirected against similar antigensexpressed in brain. (4,5) The synthesis of the anti-Hu antibody in thebrain and the specific deposits of IgG and infiltrating lymphocytesprovide circumstantial evidence for this model.

In addition to their neurological interest these cancer patients areunique in that they make a profound immunological response to theirtumor. The mechanisms underlying this specific anti-tumor response arenot well understood. It is not because these patients uniquely expressthe Hu-antigen in their small cell lung tumors. All small cell lungtumors (so far analyzed) express the Hu antigen. There are a number ofpossible mechanisms; (a) the Hu antigen may be mutated and perceived asforeign, (b) in the individuals the expression of a brain protein inextra-neural tissue provokes an extraordinary response, (c) there is anunknown secondary contributing factor e.g., viral infection.

Irrespective of the mechanism involved, the sera of these patients(which contains high-titre anti-Hu IgG) provides a reagent to isolateand characterize human small cell lung cancer tumor antigens.

SUMMARY OF THE INVENTION

An isolated nucleic acid sequence encoding Hu paraneoplastic antigenicpolypeptide is provided by this invention. This invention also providesa purified Hu antigenic polypeptide and compositions containing thepurified Hu antigenic polypeptide.

A method of detecting an antibody associated with paraneoplastic sensoryneuronopathy (PSN) is provided by this invention. This method comprisescontacting a suitable sample with the purified Hu antigenic polypeptidelabelled with a detectable marker under conditions so as to form acomplex between the purified Hu antigenic polypeptide and the antibody,detecting the presence of any complex so formed, thereby detecting anantibody associated with paraneoplastic sensory neuronopathy.

Also provided by this invention is a method of determining whether apatient exhibiting neurological symptoms harbors a tumor expressing Huantigen, which comprises contacting a suitable tumor sample from thepatient, with the monoclonal antibody directed against Hu antigen, themonoclonal antigen being labeled with a detectable marker, undersuitable conditions so as to form a complex between the antibody and thetumor antigen, detecting the presence of any complex so formed, thepresence of complex being a positive determination that the patient hasa tumor which expresses Hu antigen.

A method of inhibiting the proliferation of neoplastic cells in apatient with small cell lung cancer also is provided by this invention.This method comprises administering to the patient an effective amountof a monoclonal antibody directed to the Hu paraneoplastic tumorantigen, the monoclonal antibody being labeled with a therapeutic agent,in an amount which is effective to inhibit the proliferation of theneoplastic cells, thereby inhibiting the proliferation of neoplasticcells in a patient exhibiting paraneoplastic sensory neuronopathy.

This invention further provides a method of imaging neoplastic cells ina patient, wherein the neoplastic cells are associated withparaneoplastic sensory neuronopathy, which comprises administering tothe patient an effective amount of a monoclonal antibody directed to Huparaneoplastic antigen, the monoclonal antibody being labelled with animaging agent, under conditions to form a complex between the monoclonalantibody and the tumor antigen, imaging any complex so formed, therebyimaging neoplastic cells in a patient.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the typical Hu antigen identified in Western Blot analysisof human cortical neurons and the epitope analysis.

FIG. 2 shows restriction enzyme map of clone pHB 1.5 and clone pHB 1.0.

FIGS. 3A and 3B show the Western Blot analysis of the fusion proteinencoded by pHB 1.5.

FIGS. 4A and 4B show the nucleotide sequence for cDNA pHB 1.5. (SequenceI.D. No. 1).

FIG. 5 shows the result of a homology search conducted in data bases.Striking homology was revealed with the drosophila proteins elav and sexlethal.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides an isolated nucleic acid sequence encoding an Huparaneoplastic antigenic polypeptide. As used herein, the term Huparaneoplastic antigenic polypeptide encompasses any amino acid sequencehaving the biological activity of an Hu antigenic protein, i.e., aprotein which may specifically form a complex with an antibody which ischaracteristic of paraneoplastic sensory neuronopathy. This antibody hasalso been called anti-Hu. This antibody, i.e., anti-Hu, ischaracteristically found in patients with paraneoplastic sensoryneuronopathy, a disorder of the brain found in association withneoplasms of lung.

In one embodiment of this invention, the isolated nucleic acid sequencedescribed hereinabove is DNA. In other embodiments of this invention,the isolated nucleic acid sequence described hereinabove is cDNA, or itis RNA. In the preferred embodiment of this invention, the isolatednucleic acid sequence is a cDNA sequence as shown in sequence ID No. 1,also FIG. 4.

A vector which comprises the isolated nucleic acid molecule describedhereinabove also is provided. Suitable vectors comprise, but are notlimited to, a plasmid or a virus. This vector may be transfected into asuitable host cell to form a host vector system for the production of apolypeptide having the biological activity of the Hu antigenicpolypeptide.

This invention further provides an isolated DNA or cDNA moleculedescribed hereinabove wherein the host cell is selected from the groupconsisting of bacteria cells such as E. coli, yeast and fungi cells,insect cells and animal cells. Suitable animal cells include, but arenot limited to Vero cells, HeLa cells, Cos cells , CV1 cells and primarymouse cells.

Further provided by this invention is a method for producing apolypeptide having the biological activity of the Hu antigenicpolypeptide comprising the steps of: a) culturing the host vector systemdescribed hereinabove under suitable conditions permitting production ofthe polypeptide and b) recovering the polypeptide produced. Thisinvention also provides the polypeptide produced by this method.

Throughout this application, references to specific nucleotides are tonucleotides present on the coding strand of the nucleic acid. Thefollowing standard abbreviations are used throughout the specificationto indicate specific nucleotides:

C = cytosine A = adenosine T = thymidine G = guanosine U = uracil

This invention also encompasses DNAs and cDNAs which encode amino acidsequences which differ from those of the Hu paraneoplastic antigenicpolypeptide, but which should not produce phenotypic changes.Alternatively, this invention also encompasses DNAs and cDNAs whichhybridize to the DNA and cDNA of the subject invention. Hybridizationmethods are well known to those of ordinary skill in the art. Thisinvention also encompasses cDNA and DNA molecules characterized bychanges in non-coding regions that do not alter the phenotype of thepolypeptide produced.

Also provided by this invention is a purified, Hu antigenic polypeptide.The purified Hu antigenic polypeptide may be labeled with a detectablemarker. For the purposes of this invention, suitable detectable markersinclude, but are not limited to detectable markers selected from thegroup consisting of radioisotopes, dyes, enzymes and biotin.

This invention further provides a monoclonal antibody directed to anepitope on the Hu antigenic polypeptide. In one embodiment of thisinvention, the monoclonal antibody is a mouse monoclonal antibody. Inanother embodiment of this invention, the monoclonal antibody is a humanmonoclonal antibody.

For the isolation of mouse monoclonal antibodies, eight week old micemay be injected interperitoneally with about 50 micrograms of asynthetic, purified Hu antigenic polypeptide, (prepared as describedabove) in complete Freud's adjuvant 1:1 volume. Mice will then beboosted, at monthly intervals, with the polypeptide, mixed withincomplete Freund's adjuvant, and bled through the tail vein. On days 4,3, and 2 prior to fusion, mice will be boosted intravenously with 50micrograms of the polypeptide in saline. Splenocytes will then be fusedwith non-secreting myeloma cells according to procedures which have beendescribed and are known to those of ordinary skill in the art to whichthis invention pertains. Some time later, approximately two weeks later,hybridoma supernatant will then be screened for binding activity againstthe Hu antigenic polypeptide as described hereinafter.

Positive clones will then be isolated and propagated. Isolates of humanmonoclonal antibodies will be similar except β cells will be isolatedfrom patients and transformed with EBV. β cells will then be fused withnon-secreting myeloma cells according to procedures which have beendescribed and are known to those of ordinary skill in the art to whichthis invention pertains. Some time later, approximately two weeks later,hybridoma supernatant will then be screened for binding activity againstthe Hu antigenic polypeptide as described hereinafter. Positive cloneswill then be isolated and propagated.

In addition, this invention also provides the monoclonal antibodydescribed hereinabove conjugated to a therapeutic agent. For thepurposes of this invention, suitable therapeutic agents include, but arenot limited to, a therapeutic agent selected from the group consistingof radioisotopes, toxins, toxoids, and chemotherapeutic agents. Alsoprovided by this invention is the monoclonal antibody describedhereinabove conjugated to a detectable marker. Suitable detectablemarkers include, but are not limited to, enzymes, radioisotopes, dyesand biotin. This invention further provides monoclonal antibodies asdescribed hereinabove conjugated to an imaging agent. Suitable imagingagents include, but are not limited to radioisotopes, such as, ³²P, ³⁵S,and ¹³¹I. Methods of utilizing the monoclonal antibody conjugated to animaging agent, to image tumor cells, are well known to those of ordinaryskill in the art.

Also provided by this invention are pharmaceutical compositionscomprising the purified, Hu antigenic polypeptide described hereinabovealone, or conjugated to any one of the following, a detectable marker, atherapeutic agent, or an imaging agent, as described hereinabove and apharmaceutically acceptable carrier. Further provided are pharmaceuticalcompositions comprising the monoclonal antibody described hereinabovealone, or conjugated to any one of the following, a detectable marker, atherapeutic agent, or an imaging agent. As used herein, the term“pharmaceutically acceptable carrier” encompasses any of the standardpharmaceutical carriers, such as phosphate buffered saline solution,water, emulsions, such as a oil/water emulsions, and various types ofwetting agents.

A method of detecting an antibody associated with paraneoplastic sensoryneuronopathy “PSN”, i.e., the Hu antigenic protein, is also provided bythis invention. This method comprises contacting a suitable sample witha purified, Hu antigenic polypeptide described hereinabove underconditions so as to form a complex between the purified Hu antigenicpolypeptide and the antibody, detecting the presence of any complex soformed, thereby detecting an antibody associated with paraneoplasticsensory neuronopathy. Suitable samples include any sample suspectedcontaining an antibody associated with sensory neuronopathy, such asserum or cerebral-spinal fluid. In one embodiment of the invention thesynthetic, purified Hu antigenic polypeptide is labeled with adetectable marker selected from the group consisting of radioisotopes,dyes, enzymes and biotin. For the purposes of this invention, suitableradioisotopes include, but are not limited to, ³²P, ³⁵S, and ¹³¹I.

Also provided by this invention is a method of determining whether apatient exhibiting neurological symptoms harbors a tumor expressing theHu antigen, which comprises obtaining the suitable tumor sample from thepatient, contacting the suitable tumor sample with a monoclonal antibodydirected against the Hu antigen, under conditions so as to form acomplex between the antibody and the Hu antigen, detecting the presenceof any complex so formed, the presence of a complex being a positivedetermination that the patient harbors a tumor expressing the Huantigen. In one embodiment of this invention, the monoclonal antibody islabeled with a detectable marker. For the purposes of this invention,suitable detectable markers include, but are not limited to a detectablemarker selected from the group consisting of radioisotopes, dyes,enzymes and biotin. Suitable radioisotopes have been describedhereinabove.

Further provided by this invention is a method of inhibiting theproliferation of neoplastic cells in a patient with small cell lungcancer. This method comprises administering to the patient an effectiveamount of the monoclonal antibody or composition described hereinaboveconjugated to a therapeutic agent, in an amount which is effective toinhibit the proliferation of neoplastic cells, and under suitableconditions so as to form a complex between an antigen associated withthe neoplasm and the monoclonal antibody, thereby inhibiting theproliferation of neoplastic cells. As used herein, an effective amountis any amount which is effective to inhibit the proliferation ofneoplastic cells. As is known to those of ordinary skill in the art,effective amounts vary with the type of therapeutic agent utilized, aswell the neoplastic cell tumor being treated. It is well known to thoseof ordinary skill in the art how to determine an effective amount of asuitable therapeutic agent.

As used herein, “administering” means a method of administering to thepatient. Such methods are well known to those skilled in the art andinclude, but are not limited to administration orally, intravenously, orparenterally. Administration of the agent may be effected continuouslyor intermittently, such that the amount of the therapeutic agent in thepatient is effective to inhibit proliferation of neoplastic cells. Forthe purposes of this invention suitable therapeutic agents includeradioisotopes, toxins, toxoids, and chemotherapeutic agents.

Also provided by this invention is a method of imaging neoplastic cellsin a patient, wherein the neoplastic cells are associated withparaneoplastic sensory neuronopathy.

The method comprises administering to the patient the monoclonalantibody described hereinabove which is labelled with an imaging agent,for example ¹³¹I, or a composition containing the same, and administeredit to the patient to bind to a Hu antigen present on or within theneoplastic cells so as to form a complex between the monoclonal antibodyand the antigen, detecting any complex so formed, thereby imagingneoplastic cells in a patient having neoplastic cells expressing Huantigen. As is well known to those of ordinary skill in the art, asuitable amount of monoclonal antibody or composition is any amountwhich is effective to image the neoplastic cells, for example, fromabout 0.1 mCi to about 50.0 mCi. In addition, an effective amount of themonoclonal antibody may be an amount from about 0.01 mg to about 100 mg.Suitable methods of administering the imaging agent are as describedhereinabove.

Imaging of any complex so formed may be carried out using single photoncomputed emission tomography (SPECT) or by using a gamma camera.

This invention provides a method to isolate and clone specificanti-tumor antigen human monoclonal antibodies. The β lymphocytes willbe isolated from patient's blood, transformed with E.B.V. (Epstein BarrVirus) and selected by the specific recognition of the recombinant Huantigen.

This invention also provides a method to isolate and clone specificanti-tumor T cells. The T cells will be isolated from patient's blood,stimulated to grow with interleukin 2 and selected by specificrecognition of the recombinant Hu antigen.

Further provided by this invention is a diagnostic method useful forpredicting small cell lung cancer in patients. This method comprisescontacting a suitable sample, such as serum from the patient with alabelled antigenic polypeptide of this invention under suitableconditions so as to form a complex between the antigenic polypeptide andany antibody in the sample, detecting the presence of any complex soformed, the presence of complex being predictive of small cell lungcancer.

Materials and Methods

Materials

Sera from patients with antibody-associated paraneoplastic sensoryneuropathy was obtained from physicians. A cerebellar λ ZAP expressionlibrary was obtained from Stratagene™.

Methods

Screening of λ Cerebellar Expression Library

Recombinant phage were screened at a density of 2×10⁴ pfu per 150 mmplate of E. coli XLI-Blue. After incubation for 6 hours at 37° C., theplates were overlaid with filters soaked in 1PTG (10 mM) and incubatedfor a further 12 hours at 37° C. The filters were then removed andincubated with anti Hu sera (2 μg/ml IgG), for 2 hours at roomtemperature. The filters were then washed with TBST (50 mM Tris (pH7.4), 100 mM NaCl, 0.2% Triton) and incubated with I¹²⁵ Protein A. Afterwashing with TBST the filters were exposed to XRA5 film at −70° C.Clones yielding positive signals were purified by several rounds ofantibody screening until 100% of the plaques gave positive signals.

Analysis of Fusion Proteins

Phage clones were subcloned into p Bluescript (pBs) using the phagerescue protocol. [10] Individual clones were grown to an optical densityof 0.6 and induced by adding IPTG (10 mM). After 1 hour of induction at37° C., the bacterial cells were isolated by centrifugation resuspendedin lysis buffer (2% SDS, 50 mM Tris (pH 6.8)). Lysates were thenresolved by 8% polyacrylamide SDS gel electrophoresis and transferred tonitrocellulose [11]. The filters were then incubated with anti-Hu sera(5 μg/ml in TBST) for 2 hours at room temperature. The filters were thenwashed with TBST and incubated with I¹²⁵ protein A (0.1 μCi/ml). After afurther washing with TBST, the filters were exposed to XRA5 film at −70°C.

DNA Sequence Analysis

All sequencing was based on the dideoxy termination method [10]. Doublestrand DNA was sequenced on both strands using SK, KS, M13 universal andreverse primers, and internal oligonucleotide primers. Sequences weremerged and analyzed for open reading frame and functional motifs withthe Macvector analysis software.

Results

Isolation of Positive λ Clones

Screening of the cerebellar cell λ ZAP expression library with a typicalhigh-titer anti-Hu sera resulted in the isolation of 8 positive clones.None of the clones we recognized by normal human sera. The clones werecharacterized by insert size and similarity (by Southern blothybridization) to each other. Clones HB 1.5 and HB 0.8 belong to onegene product. The relationship between the human cerebral antigen Hu andthe fusion protein encoded by clones HB 1.5 and HB 0.8 was examined bythe epitope selection method. In this procedure the antibodies thatrecognize the recombinant fusion protein are isolated and reacted with aWestern blot of the tissue antigen. Purified antibodies from bona-fideclones should identify the original antigen of interest. FIG. 1 (lane 2)shows the typical Hu antigen identified in western blot analysis ofhuman cortical neurons. Normal human serum only identifies anon-specific antigen of 12 kb in wt. FIG. 1 (lane 4) shows that affinitypurified antibodies selected by reactivity with the fusion proteinsencoded by clones HB 1.5 recognized the Hu-antigen expressed in humancortical neurons. A mock purification of anti-Hu sera employing anirrelevant fusion protein served as the negative control (FIG. 1, lane3). It was therefore concluded that the gene product encoded by clonesHB 1.5 and HB 0.8 was related to the Hu-antigen.

Both λ phage clones were then subcloned into PBS utilizing the phagemidrescue procedure. The resulting bacterial plasmids pHB 1.5 and pHB 0.8had inserts of 1.5 kb and 0.8 kb, respectively. Restriction enzymedigestion confirmed that the two clones were related and a simplifiedmap is shown in FIG. 2.

Specific Recognition of cDNA Clones by Anti-Hu Sera

The reactivity of anti-Hu sera and various negative control sera wasestablished by Western blot analysis of the fusion protein encoded bypHB 1.5. Anti-Hu sera identify a fusion protein of 55 kd in extracts ofpHB 1.5 (FIG. 3A, lane 4). No reactivity was observed with extracts ofPBS (the parental plasmid vector with no insert) (FIG. 3A, lane 3). Noreaction of the pHB 1.5 fusion protein was observed with normal humanserum (FIG. 3A, lane 3). The minor bands visible in FIG. 3A (lane 4)correspond to proteolytic fragments of the 55 kd fusion protein. FIG. 3Bshows that reactivity with sera previously characterized as anti-Hu werepositive (lanes 3-8). Negative controls included: normal human sera(lanes 1 and 2), sera from patients with antibody-associatedparaneoplastic cerebellar degeneration (anti-yo), serum from anotherantibody-associated paraneoplastic syndrome (anti-Ri), and lupus serum.In total, all fifty available anti-Hu sera were positive and noreactivity was detected with a total of 150 negative sera. It wastherefore concluded that the reactivity of serum with the pHB 1.5 fusionprotein is a reliable assay for antibody-associated paraneoplasticsensory neuronopathy (anti-Hu syndrome).

Histochemical Identification Of Lymphocytes That Recognize the Huantigen

The brain and small cell lung cancer of patients suffering from theanti-Hu syndrome contain infiltrating lymphocytes. In order to establishwhether the infiltrating lymphocytes are directed against the Huantigen, the fusion protein encoded pHB 1.5 was purified by preparativegel electrophoresis and labelled with biotin. Protein eluted from thesera region of a preparative gel containing E. coli extract was alsobiotinylated and served as a negative control. Biotinylated recombinantHu-antigen and irrelevant E. coli protein were then incubated withfrozen sections of brain and small cell lung cancer tissue from ananti-Hu patient. Bound protein was then visualized by incubation withavidin biotin peroxidase complex and DAB. Biotinylated HuD antigenreacted specifically with lymphocytes in the small cell lung cancer andbrain of an affected individual. There was no reactivity with normalhuman brain. The irrelevant E. coli protein did not specifically reactwith any cell line in any of the tissues. Examination of the stainedlymphocytes revealed them to be both B and T cells.

Sequence Analysis of pHB 1.5 and pHB 0.8

pHB 1.5 and pHB 0.8 use sequenced completely on both strands (FIG. 2).There is a poly (A) tail at the 3′ end of pHB 1.5. A long open readingframe encoding a predicted bas c (P.I.=9.5) protein of 380 amino acids(M.Wt=41 kd) was found starting with an ATG at position 95 andterminating with a codon at position 1234. The open reading frame ispreceded by three in frame termination codons at positions 5, 8, and 17indicating that the ATG at 95 which satisfies the Kozak consensus ruleis probably the true initiating ATG. Examination of the rare codon usagealso indicated that the sequences preceding the ATG at 95 do not codefor protein. The protein encoded by pHB 1.5 is not in frame with the βgalactoside AUG.

The predicted amino acid sequence of HuD was used in a homology searchof data bases and revealed striking homology with the drosophila proteinelav and sex lethal. (FIG. 5A and 5B).

References

1. Henson, R. and Urich, H. “Cancer and the nervous system.” 1982Blackwell Scientific. Oxford.

2. Graus, F., Cordon-Cardo, C. and Posner, J. Neuronal antinuclearantibody in sensory neuronopathy from lung cancer. Neurology 35:538-543,1985.

3. Dalmau, J., Furneaux, M. M., Gralla, R. J., Kris, M. G., Posner, J.B. Detection of the anti-Hu antibody in the serum of patients with smallcell lung cancer-a quantitative western blot analysis. Ann. Neurol27:544-552, 1990.

4. Furneaux, H., Reich, L. and Posner, J. Central nervous systemsynthesis of autoantibodies in paraneoplastic syndromes. Neurology. 40:1085-1091, 1990.

5. Dalmau, J. et al. Immunological Study of Central Nervous System andTumor in Tumor Paraneoplastic Encephalomyelitis/Sensory NeuronopathyAssociated With Small Lung Cancer, Ann. of Neur., submitted forpublication.

6. Brain, L. and Wilkinson, M. 88: 465-478, 1965.

7. Furneaux, H. and Posner, J. Paraneoplastic neurological syndromes.Proc. Assoc. Res. Nerv. Men. Dis. 68: 187-219, 1990.

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 1(2) INFORMATION FOR SEQ ID NO:1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 1469 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO     (vi) ORIGINAL SOURCE:          (A) ORGANISM: cDNA enco #ding Hu antigenic protein   (vii) IMMEDIATE SOURCE:           (B) CLONE: pHB 1.5     (x) PUBLICATION INFORMATION:           (A) AUTHORS: Furneaux, #Henry M.           (C) JOURNAL: Cell    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:CCAATAGTAG TCATTTTAAA TATATATTCT GAAATCTTTG CAAATTTTAA CA#GAAGAGTC     60GAAGCTCTGC GAGACCCAAT ATTTGCCAAT AAGAATGGTT ATGATAATTA GC#ACCATGGA    120GCCTCAGGTG TCAAATGGTC CGACATCCAA TACAAGCAAT GGACCCTCCA GC#AACAACAG    180AAACTGTCCT TCTCCCATGC AAACAGGGGC AACCACAGAT GACAGCAAAA CC#AACCTCAT    240CGTCAACTAT TTACCCCAGA ATATGACCCA AGAAGAATTC AGGAGTCTCT TC#GGGAGCAT    300TGGTGAAATA GAATCCTGCA AACTTGTGAG AGACAAAATT ACAGGACAGA GT#TTAGGGTA    360TGGATTTGTT AACTATATTG ATCCAAAGGA TGCAGAGAAA GCCATCAACA CT#TTAAATGG    420ACTCAGACTC CAGACCAAAA CCATAAAGGT CTCATATGCC CGTCCGAGCT CT#GCCTCAAT    480CAGGGATGCT AACCTCTATG TTAGCGGCCT TCCCAAAACC ATGACCCAGA AG#GAACTGGA    540GCAACTTTTC TCGCAATACG GCCGTATCAT CACCTCACGA ATCCTGGTTG AT#CAAGTCAC    600AGGAGTGTCC AGAGGGGTGG GATTCATCCG CTTTGATAAG AGGATTGAGG CA#GAAGAAGC    660CATCAAAGGG CTGAATGGCC AGAAGCCCAG CGGTGCTACG GAACCGATTA CT#GTGAAGTT    720TGCCAACAAC CCCAGCCAGA AGTCCAGCCA GGCCCTGCTC TCCCAGCTCT AC#CAGTCCCC    780TAACCGGCGC TACCCAGGTC CACTTCACCA CCAGGCTCAG AGGTTCAGGC TG#GACAATTT    840GCTTAATATG GCCTATGGCG TAAAGAGACT GATGTCTGGA CCAGTCCCCC CT#TCTGCTTG    900TTCCCCCAGG TTCTCCCCAA TTACCATTGA TGGAATGACA AGCCTTGTGG GA#ATGAACAT    960CCCTGGTCAC ACAGGAACTG GGTGGTGCAT CTTTGTCTAC AACCTGTCCC CC#GATTCCGA   1020TGAGAGTGTC CTCTGGCAGC TCTTTGGCCC CTTTGGAGCA GTGAACAACG TA#AAGGTGAT   1080TCGTGACTTC AACACCAACA AGTGCAAGGG ATTCGGCTTT GTCACCATGA CC#AACTATGA   1140TGAGGCGGCC ATGGCCATCG CCAGCCTCAA CGGGTACCGC CTGGGAGACA GA#GTGTTGCA   1200AGTTTCCTTT AAAACCAACA AAGCCCACAA GTCCTGAATT TCCCATTCTT AC#TTACTAAA   1260ATATATATAG AAATATATAC GAACAAAACA CACGCGCGCA CACACACACA TA#CACGAAAG   1320AGAGAGAAAC AAACTTTTCA AGGCTTATAT TCAACCATGG ACTTTATAAG CC#AGTGTTGC   1380CTAGTATTAA AACATTGGGT TATCCTGAGG TGTACCAGGA AAGGATTATA AT#GCTTAGAA   1440 AAAAAAAAAG AAAAAAAAAA AACAAAAAA         #                   #          1469

What is claimed is:
 1. A method of detecting an antibody associated withparaneoplastic sensory neuronopathy in a patient sample, comprisingcontacting the sample with a purified Hu antigenic polypeptide underconditions effective to form a complex between the purified Hu antigenicpolypeptide and the antibody, detecting the presence of any complex soformed, thereby detecting the antibody associated with paraneoplasticsensory neuronopathy.
 2. The method of claim 1, wherein the purified Huantigenic polypeptide is labelled with a detectable marker.
 3. Themethod of claim 2, wherein the detectable marker is selected from thegroup consisting of a radioisotope, a dye, an enzyme and biotin.
 4. Amethod of determining whether a patient exhibiting neurological symptomsharbors a tumor expressing Hu antigen, comprising contacting a samplefrom the patient with an antibody directed to an epitope on a purifiedHu antigenic polypeptide.
 5. The method of claim 4, wherein the antibodyis labelled with a detectable marker.
 6. The method of claim 5, whereinthe detectable marker is selected from the group consisting of aradioisotope, a dye, an enzyme and biotin.
 7. A method of inhibiting theproliferation of neoplastic cells in a patient having paraneoplasticsensory neuronopathy which comprises administering to the patient aneffective amount of of an antibody directed to an epitope on purified Huantigenic polypeptide, wherein the antibody is conjugated to atherapeutic agent, effective to inhibit the proliferation of neoplasticcells in the patient having paraneoplastic sensory neuronopathy.
 8. Amethod of claim 7, wherein the therapeutic agent is a radioisotope,toxin, toxoid or chemotherapeutic agent.
 9. The method of claim 1,wherein said Hu polypeptide is purified by gel electrophoresis.
 10. Themethod of claim 9, wherein said gel electrophoresis is carried out onbrain tissue containing Hu polypeptide.
 11. The method of claim 1,wherein said patient sample is a blood sample.